Gen Pharmac Vol 23, No 6, pp 109%1102, 1992

0306-3623/92 $5 00 + 0 00 Copyright © 1992 Pergamon Press Ltd

Pnnted m Great Britain All rights reserved

I N H I B I T I O N IN L-TYPE Ca 2+ C H A N N E L BY S T I M U L A T I O N OF P R O T E I N K I N A S E C IN I S O L A T E D G U I N E A PIG V E N T R I C U L A R C A R D I O M Y O C Y T E S HIROYASU SATOH Department of Pharmacology, Nara Medical Umverslty, 840 Shljo-cho, Kashihara, Nara 634, Japan (Recewed 7 Aprd 1992)

Abstract--1 Electrophyslologmal effects of phorbol esters on the L-type Ca 2+ current (lc~(L))In isolated single ventncular cells from guinea pig hearts were mvestlgated 2 In whole-cell voltage-clamped myocytes, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) at 10-7 M mlubtted IC~(L) An antagonist of protein kinase C (PK-C), H-7, at 10-5 M did not modify the TPA-mduced mhlbmon The time-course of mactwauon process for Icazt) was greatly slowed 3 In cell-attached patch-clamp experiments, TPA (10 -~ M) also markedly decreased the opening of L-type Ca 2+ channels The conductance was unaffected 4 Even H-7 (10 -5 M) alone inhibited the opening of the channels Addmon of TPA (10-7-10 -s M) caused further decrease m the opemng 5 On the other hand, 4-~-phorbol-12,13-dldecanoate (not a PK-C activator) had no effect on the Ca 2+ channels 6 These results mdzcate that the PK-C activation induced by TPA greatly depresses the opemng of L-type Ca 2+ channels m ventncular cell membranes

INTRODUCTION

Protem l~nase C (PK-C), Ca 2+- and phosphohplddependent protein kmase, appears to be an important component of membrane signal transductlon for a variety of blologmal actwators of substances (Nlshmuka, 1984) There have already been reports on physmlogmal effects reduced by P K - C stlmulaUon, whtch (1) causes mcreases m R N A , protem and D N A synthesis (Bmrd et a l , 1971, W a n g et a l , 1975, Brown et a l , 1979), (2) causes platelet aggregatmn and phosphorylatton of a pepttde m blood platelets (Chmng et al , 1981, Whtte et al , 1974, Carrol et al , 1982), (3) enhances N a ÷ entry mto 3T3 cells ( N a + - H + exchange)(Dmker and Rozengurt, 1981), and (4) increases N a + - K + pump a c t m t y (Dicker and Rozengurt, 1981, Moroney et a l , 1978) In addmon, Satoh and Hashlmoto (1989) have shown that P K - C actwatlon by phorbol esters produced cellular calcmm overload m rabbit smo-atnal (SA) node cells A sUmulant specifically brads to a cell surface receptor, which actwates a phosphohpase C for phosphatldyl-momol (PI) located on the cytosohc face o f the plasma membrane The actwe enzyme releases phosphatldyl-momol-4,5-blphosphate (PIPe) into the cytosol and leaves m y o - m o m o l - l , 4 , 5 - t n p h o s p h a t e (IP3) and dmcylglycerol (DG) m the membrane IP3 mobthzes Ca 2+ stored m the sarcoplasnuc ret~culum (SR) (Berndge, 1986, Wflhamson et a l , 1985) D G acuvates P K - C by stablhzmg Rs msertmn into the membrane Phorbol esters possess a DG-hke structure Actavated P K - C then phospborylates substrate proteins and thereby the physmloglcal responses would be ehcRed (Streb et a l , 1983, Bemdge, 1986) TPA, a sUmulator of P K - C (12-O-tetradecanoylphorbol-13-acetate), had a small increase m the OP 23/~--L

L-type Ca 2+ current (/Ca(L)) m rabbit SA node cells (Satoh and Hashlmoto, 1989) But the effects of P K - C actwaUon were controverstal Thus, tn the present expenments, I sought to examine whether T P A also sUmulates the L-type Ca 2+ channels m single ventncular muscle cells To confirm whether the effects reduced by T P A are due to actwaUon of PK-C, the effects were compared wtth those of P D D (which lacks an actwator of PK-C), and H-7 (an inhibitor of PK-C) was used MATERIALS AND METHODS

Cell preparatton

Guinea pigs eRher sex, wetglung 250-400 g, were anesthetized with mtrapentoneal reJection of sochum pentobarbital (30 mg/kg) Under arhficlal ventalataon, the chest was opened and the aorta was cannulated m suu The heart was then dissected out and was perfused wRh normal Tyrode solution on the langendorff apparatus After the blood was completely washed out, the heart was perfused voth Ca2+-free Tyrod¢ solution, and the spontaneously beating heart was ceased Then, the perfusate was swRched to low-Ca2+ (30-60#M) Tyrode solution contmnmg 0 4 mg/ml collagenase (Type I, Sigma Chemical Co, St Louis, Mo ) for about 20 mm After the collagenase m the heart was washed out by hlgh-K + and low-Cl- solutmn (KB solution), the heart was dissected wRh scissors The final cell suspenston was premcubated in the KB soluUon for at least 60 mm at 4°C prior to the experiments The temperature of all solutions was maintained at 36 5°C Whole-cell voltage clamp recording

Whole-cell patch clamp experiments (Hamfll et al, 1981, Satoh and Sperelakis, 1992) were done using glass patch pipettes The t~p diameter was 3-5/~m and the resmtance was 3-10 Mf~ The membrane capacRance was 130 ± 21 pF (n = 24) Current and voltage records were stored on magnetic tape for computer analysts (NEC 98 series) The

1097

1098

HIROYASUSATOH @ Control

ooA

0

TPA 10 -7 M

&

+H-7

10-5M

0

I 100 msec

Fig 1 Inhlbmon of L-type C a 2+ current by phorbol ester m single ventncular muscle cell Whole-cell voltage clamp expenment was exerted Test pulses (for 500 msec) were apphed to 0 mV from holding potentml of - 3 0 mV Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) was added to the bath solutmn Symbols used are control (O), TPA 10 -7 M ((3) and TPA plus H-7 10-5 M (A) Short hne at the left of superimposed current records represents zero current level composmon of the mo&fied Tyrode solutmn was as follows (raM), NaC1 137, KC1 5 4, CaCI2 1 8, MgCI2 0 5, NaH2PO4 0 33, glucose 5 0, Hepes 5 0 (pH 7 4) The pipette (mtracellular) solutmn contained (mM), CsOH 110, aspartlc acad 90, CsC1 20, K2ATP 5, Creatme phosphate 5, EGTA 5, MgC12 1, Hepes 5 (N-2-hydroxyethylplpera2ane-N'-2-ethanesulfome acad, Wako Pure Chenucal Industries, Osaka, Japan) and cychc AMP 0 05 Intracellular pH was adjusted to 7 4 and pCa was 7 2 The values m text are represented as mean + SEM Umtary current recording The cell-attached patch-clamp techmque was performed according to the methods of HanuU et al (1981) The resistance of the patch electrode was 3-5 Mf~, and the tip of the electrode was coated with Sylgard (KE106, Shm-etsu Chenucal Co ) Umtary current traces were stored on a video recorder (BR6400, Victor) using a PCM converter system (RP-880, NF Electromc Clrcmt Design, Tokyo) The data were analyzed on an Hltach! E600 computer Current traces were filtered with a cut-off frequency of 1 kHz for plotting (FV-625, NF) The membrane potentml of the patch was calculated as the difference between the restang potentml and the ptpette potentml For the single-channel recording, the pipette solution contained (raM) BaCI: 50, chohn-C170, glucose 5, Hepes 0 5, Bay K 86442 10 -6 M and tetrodotoxm 10-5 M (pH 7 4) All experiments were performed at 37°C Drugs Drugs used m this study were 12-O-tetradecanoylphorbol-13-acetate (TPA) and 4-~-phorbol-12,13-dldecanoate (PDD), (Sigma Chenucal Co, St Lores, Mo ) Both phorbol esters were &ssolved m &methyl sulforade (DMSO), which was &luted 7000-20,000 ttmes m the perfusmn medmm, and stocked at - 10°C Also, H-7 [1-(5-1soqmnohnylsulfonyl)-2methylplperazme dlhydrochlonde] was &ssolved m dlslalled water Since solutmn m the bath was exchanged within I nun and the effects of drugs completely reached a steady state wltlun 3 nun, the data were obtained for 3-5 nun after changing to the new solutmn

RESULTS L-type Ca e+ current Effects of T P A on the L-type Ca 2+ current (Ic.(L)) m isolated guinea pig ventncular myocytes were examined by whole-cell patch-clamp techmque (Fig 1) Holding potential was - 3 0 mV Test pulses were apphed to 0 mV for 500 msec duraUon T P A (10 -7 M), a P K - C actwator, was added to the bath solution At 6 nun after apphcatlon, T P A depressed the IC~(L) current The summarized percentage mhlb m o n were 21 2 + 2 8% (n = 10, P < 0 0 0 1 ) The time-course of mactwatlon process for (Ic~L)) was greatly slowed in the presence of T P A (10-7M) Further a d d m o n of H-7 (10 -s M) (an mhtbltor of PK-C) had httle or no effect on the depressant IC~L) m all of 7 cells Single L-type Ca 2+ channels To examine the effects of P K - C actwataon reduced by T P A on umtary L-type Ca 2+ current, cell-attached patch clamp experiments were performed Depolarr a n g test pulses of 70 mV for 300 msee were apphed from the resting potential ( - 8 0 mV) As shown m Fig 2, T P A (10 -7 M) reduced the opening probablhty by 28 4 + 5 1% (n = 6, P < 0 001) In another cell, H-7 (10 -5 M) alone (an antagomst o f PK-C) slgmficantly dad not affect the opemng probablhty, but tended to decrease it (by 6 7__.20%, n = 6 , P > 0 0 5 ) (Figs 3A and B) A d d m o n of T P A (10-SM) decreased the opening by 3 7 2 + 4 1% (n = 6, P < 0 01) (Fig 3C) Increasing concentratmn of T P A to 10 -7 M inhibited it by 58 2 __. 3 8% (n = 6, P < 0 001) (F~g 3D) N o antagomstm aetmn of H-7 was observed m all of 6 cells (n = 6) On the other hand, P D D (10 -7 M) (not an aetwator of PK-C) &d not cause any effect of the channel opening (Fig 4)

IntubRmn in L-type Ca 2+ channel

1099

TPA 10 -7 M

Control

70 mV Depo

"e~mr~-" ~

....

:-" "~h-""-" ~ = t q . * ¢ " " ' ~ ¢ ~

50 msec Fig 2 Depression m the opemng of L-type Ca 2+ channel m the presence of TPA Cell-attached patch clamp experiments were performed using a single gmnea pig ventncular carchomyocyte Umtary channel opemngs are shown by the steps to the reward &rectmn Depolanzang test pulses of 70 mV for 300 msec were apphed from the resting potvntml ( - 8 0 mV) Pipette solutmn included Ba'+ (50 raM) and Bay K 8644 (10-6M) TTX (10-SM) was added to the bath solutmn

(A)

Control

(B)

H - 7 10 -5 M 70 mV Depo

(C)

+ TPA 10 -8 M

(D)

+ TPA 10 -7 M

50 m sec

Fig 3 Effects of H-7 and H-7 plus TPA on the L-type Ca 2+ channel. Cell-attached patch clamp experiments were performed Depulanzmg test pulses of 70 mV for 300 msec were apphed from the rcsUng potential ( - 8 0 mV) pipette soluuon included Ba 2+ (50 raM) and Bay K 8644 (10 -6 M) TTX (10 -5 M) was added to the bath solution (A) Current traces of umtary L-type Ca '+ channel (B) Umtary current traces m H-7 (10-s M) (C and D) Umtary current traces m H-7 and TPA (10-SM and 10-TM)

1100

HmoY~u SATOH Control

PDD 10-;' M 70 mV Depo

I50 rnsec

Fig 4 No effect of PDD on the L-type Ca 2+ channels in a single guinea pig ventncular muscle cell Non-activator of PK-C among phorbol ester analogues, 4-~-phorbol-12,13-dldecanoate (PDD, 10 -7 M), was added to the bath solutmn Depolarizing test pulses of 70 mV for 300 msec were apphed from the resting potentml (-80mV) lhpette solutmn included Ba2+ (50 mM) and Bay K 8644 (10-6 M) T r x (10 -5 M) was added to the bath solutmn DISCUSSION Phorbol esters may substitute for dlacylglycerol (DG) and permanently actwate protein kmase C Phorbol esters play a role as tumor promoters, and would cause many physmloglcal responses (see the Introductmn sectmn) Recent kmehc analyses have indicated that TPA, which possesses a DG-hke structure, can substitute for D G at extremely low concentrations (10 -1° M) (Nlshlzuka, 1984, Ashendel, 1985) and also that Rs primary sRe of actmn Is the cell surface (Wemstem et al, 1979, Blumberg, 1980) Two types (L and T) of Ca 2+ channels have been reported to be present m the cardmc muscle cells (Nflms et al, 1985, Hofmann et al, 1987) These are dlstmgmshed by differences m (1) their voltagedependency of actlvatmn and reactivation, (2) their sensmvlty to dlhydropyndme blockers, (3) their single-channel conductance, and (4) their permeabflRy to various divalent catmns In this study, the characteristics of the L-type Ca 2÷ current (/ca(L)) were investigated The effects of phorbol esters on /Ca(L)In cardmc and vascular smooth muscle cells and m neurons are still controversml The actwatmn of PK-C by TPA enhanced the voltage-dependent Ca 2+ current m bag cell neurones of the mollusc Aplysta (DeRaemer et al, 1985), whereas TPA attenuates IC~L) m sensory neurones (Rane and Dunlap, 1986) In cardmc cells (rabbit SA node and neonatal rat heart), TPA had a small mcrease m IC~(L)(Dosemecl et al, 1988, Satoh and Hashlmoto, 1989) In smooth muscles (frog wscera and rat aorta), phorbol esters enhanced Ic.(L) (Vlvaudou et al, 1988, Fish et al, 1988) Furthermore, m cultured vascular smooth muscles (A7r5 cell hne), TPA shghtly enhanced IC.tL) m almost half of all cells, and mlublted/ca(L) m the half cells (Satoh and Sperelakls, 1992) In the present

experiments (guinea pig ventncular cardlomyocytes), whole-cell and patch-clamp experaments clearly showed that TPA inhibited the amphtude of IC~(L)and the opemng of L-type Ca 2÷ channels And PDD (non-actwator of PK-C) did not affect the opening Therefore, these results mdlcate that these lnMbmons of/Ca(L) and the opening probability are due to the PK-C actwatlon The mhlbmon was not modified by H-7 of PK-C inhibitor It has been shown that H-7 mhlbRs three different protein kmases with equal actmty, having K~ values of 3 - 6 / t M for cychc nucleotldes (cAMP and cGMP)-dependent klnases (PK-A and PK-G) as well as for PK-C, but it ~s less actwe as an mhlbRor of myosin hght chain kmase (K,= 97/tM) (Garland et al, 1987) Thus, It seems that the small decrease (by about 7% m this study) m the opening of Ca 2÷ channels reduced by H-7 may be due to the mlubmon of PK-A These results indicate that H-7 has no or httle inhibitory action of PK-C actwatmn, and the mhlblUon of the channel openings is also nonspecific Phorbol esters can elevate the cellular Ca 2+ concentration ([CAD (Satoh and HasMmoto, 1989) Because phorbol esters ehclted the cellular calcmm overload (dysrhythmm) m spontaneously beating SA node cells Sugden et al (1985) reported that the phorbol esters potentmted fl-adrenergm stimulation of cychc A M P accumulation m the presence of lsoproterenol In addmon, it has been demonstrated, by the experiments using fura-2 (a fluorescent calcmm indicator) m my laboratory (but unpubhshed data), that actually, phorbol esters elevated [Ca]. In the present expenments, however, TPA mhlbRed lcaL) and decreased the opemng probabdlty, slgmficantly Therefore, these results suggest that PK-C actwatlon by the phorbol esters might elevate [Ca], due to other

Intubmon m L-type Ca 2+ channel complex mtracellular events [1 e release from Ca 2+ stores (SR and mltochondna), blockade of Ca 2+ re-uptake, blockade o f Ca 2÷ pump and feedback of PI turnover, etc ] Further experiments are reqmred to elucidate th~s SUMMARY

1 Electrophyslologlcal effects of phorbol esters (sUmulators of protem kmase C) on the L-type Ca :+ current (/Ca(L)) m Isolated single ventncular cells from gumeaplg hearts were mvesUgated by means of whole-cell and patch voltage-clamp techmques 2 In whole-cell voltage-clamped myocytes, 12-0tetradecanoylphorbol- 13-acetate (TPA) at 1 0 - 7 M inhibited the (/Ca(L)) current by 21 2 + 2 8% (n = 10, P < 0 001) Test pulses were apphed to 0 m V from holding potentml of - 3 0 mV An antagomst of protein kanase C, H-7 [1-(5-1soqumohnylsulfonyl)-2-methylplpera n n e dlhydrochlonde], at 10-SM did not modify the TPA-lnduced lnhlbmon The t i m e course of inactivation process for /Ca(L) was greatly slowed 3 In cell-attached patch-clamp experiments, T P A (10 -7 M) also markedly decreased the opening of L-type Ca 2÷ channels by depolarizing pulses of 7 0 m V Ba 2÷ ( 5 0 m M ) and Bay K8644 (10 -s M) were added to the pipette solution to faclhtate the measunng of the channel opening The conductances were unaffected, 22 + 3 pS m the absence and 2 1 _ 2 pS in the presence of T P A (10 -7 M) 4 Even H-7 (10 -5 M) alone inhibited the openmg of the channels (by 6 7 + 2 0 % , n=6, P > 0 05) AddlUon of T P A (10 -s M) decreased the opening by 3 7 2 + 4 1% (n = 6 , P < 0 01), and at 10 -7 M, T P A inhibited it by 58 2 + 3 8% (n=6, P